A kind of terlipressin sustained-release microsphere and preparation method thereof

Abstract

The invention relates to a terlipressin sustained-release microsphere and a preparation method thereof. The method comprises the following steps: S1, dissolving terlipressin acetate in pure water to obtain an inner water phase; S2, dissolving polylactic acid-glycolic acid copolymer in an organic solvent to obtain an oil phase; S3, dissolving polyvinyl alcohol in pure water, and adjusting the pH value to 7.8-8.5 to obtain an outer water phase; S4, adding the inner water phase to the oil phase, emulsifying, and obtaining W / O colostrum; S5, mixing the colostrum with the outer water phase, performing secondary emulsification, and obtaining a W / O / W double emulsion; S6, stirring to remove the organic solvent at room temperature, centrifugally collecting the microspheres, washing with pure water, and then freeze-drying to obtain a freeze-dried product of terlipressin sustained-release microspheres. Based on the alkaline isoelectric point of terlipressin, the present invention reduces the solubility of terlipressin in the external aqueous phase by adjusting the pH of the external aqueous phase to 7.8-8.5, avoids the migration of terlipressin from the internal aqueous phase, and effectively improves the encapsulation rate of terlipressin microspheres.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a terlipressin sustained-release microsphere and a preparation method thereof. Background Art

[0002] Terlipressin, chemically known as triglycyllysine vasopressin, is an artificially synthesized analog of endogenous vasopressin. It is a prodrug that, after entering the body, is removed by aminopeptidase and its three glycyl residues at the N-terminus are removed, slowly releasing pharmacologically active lysine vasopressin. The main function of terlipressin is to contract visceral vascular smooth muscle, reduce visceral blood flow (such as reducing blood flow in the mesentery, spleen, uterus, etc.), thereby reducing portal vein blood flow and portal vein pressure. It can also act on smooth muscles such as the esophagus and uterus; on the other hand, terlipressin can also reduce the effect of plasma renin concentration, thereby increasing renal blood flow in patients with hepatorenal syndrome, improving renal function and increasing urine output. Terlipressin has been proven to have a very significant effect in complications of portal hypertension in cirrhosis (such as esophageal varicose bleeding, hepatorenal syndrome, ascites, etc.), and is also widely used in other fields such as septic shock, liver resection / transplantation, acute liver failure, cardiac arrest, etc.

[0003] The current dosage form of terlipressin is injection, with slow intravenous push or continuous drip as the main method of administration. In the treatment of portal varicose bleeding, the drug needs to be administered every 4 to 6 hours, and it is recommended to continue to be administered for 3 to 5 days. Multiple administrations during treatment cause inconvenience to patients. Clinical evidence shows that the use of continuous slow administration can reduce the risk of serious side effects of terlipressin. However, there are currently no terlipressin sustained-release preparations in clinical practice. Therefore, the development of terlipressin microsphere preparations is of great significance to fill the gap in clinical medication, enrich clinical medication options, and simplify clinical medication methods.

[0004] The W / O / W emulsion method is one of the most commonly used methods for preparing peptide drug microsphere preparations. The colostrum is formed by adding the organic phase to the inner aqueous phase for emulsification, and then the colostrum is added to the outer aqueous phase to form a double emulsion. Since peptide drugs are easily soluble in water, the addition of a large amount of outer aqueous phase can easily cause the migration of peptide drugs from the inner aqueous phase, resulting in a decrease in the encapsulation rate of the microspheres and a burst release phenomenon. Therefore, how to improve the encapsulation rate and stability of water-soluble peptides has become a key issue for the successful application of microspheres in the drug delivery of peptide substances. Summary of the invention

[0005] The technical problem to be solved by the present invention is: the present invention provides a terlipressin sustained-release microsphere and a preparation method thereof, which can effectively improve the encapsulation rate and sustained-release effect of the terlipressin microsphere, and has a reference significance for the development of terlipressin microsphere preparations.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0007] In one aspect, the present invention provides a method for preparing terlipressin sustained-release microspheres, comprising the following steps:

[0008] S1, dissolving terlipressin acetate in pure water to prepare an inner water phase;

[0009] S2, dissolving the polylactic acid-glycolic acid copolymer in an organic solvent to obtain an oil phase;

[0010] S3, dissolving polyvinyl alcohol in pure water and adjusting the pH value to 7.8-8.5 to prepare an external water phase;

[0011] S4, adding the inner water phase to the oil phase for emulsification to obtain W / O colostrum;

[0012] S5, mixing the colostrum with the external water phase, performing secondary emulsification, and obtaining a W / O / W double emulsion;

[0013] S6. Stirring at room temperature to remove the organic solvent, collecting the microspheres by centrifugation, washing with pure water, and then freeze-drying to obtain the freeze-dried terlipressin sustained-release microspheres.

[0014] The invention provides a method for preparing terlipressin sustained-release microspheres, which adopts a W / O / W double emulsion solvent volatilization method to prepare terlipressin sustained-release microspheres. The method is based on the alkaline isoelectric point of terlipressin, and reduces the solubility of terlipressin in the external aqueous phase by adjusting the pH of the external aqueous phase to 7.8-8.5, thereby avoiding the migration of terlipressin from the internal aqueous phase, effectively improving the encapsulation rate of the terlipressin microspheres, and having the characteristics of less solvent residue, uniform particle size distribution, good sustained-release effect, etc.

[0015] Optionally, in step S1, the mass ratio of the terlipressin acetate to the pure water is 1:1 to 1:3.

[0016] Optionally, step S1 further includes adding a protein protective agent to the inner aqueous phase, wherein the protein protective agent is at least one of sucrose, fructose, maltose and trehalose, and the concentration thereof is 10 to 30 mg / mL.

[0017] Optionally, in step S2, the mass ratio of the polylactic acid-glycolic acid copolymer to the organic solvent is 1:2-1:3, the monomer ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is 50:50, and the organic solvent is dichloromethane.

[0018] Optionally, in step S3, the external aqueous phase contains 0.1 wt % polyvinyl alcohol;

[0019] The pH value range is adjusted by a pH adjuster, and the pH adjuster is sodium bicarbonate and sodium hydroxide.

[0020] Optionally, in step S4, the volume ratio of the inner water phase to the oil phase is 1:10 to 1:20;

[0021] The emulsification adopts a high-speed homogenization emulsification method, the emulsification temperature is 20-35° C., the emulsification speed is 15000 rpm, and the emulsification time is 10-20 minutes.

[0022] Optionally, in step S5, the volume ratio of the colostrum to the external aqueous phase is 1:100 to 1:500;

[0023] The secondary emulsification adopts a high-speed homogenization emulsification method, and the secondary emulsification rotation speed is 10000-18000rpm.

[0024] Optionally, step S6 further comprises, before the freeze-drying treatment, stirring and adding a freeze-drying protective agent to the microsphere concentrate obtained after washing to prepare a microsphere suspension, wherein the freeze-drying protective agent is a mannitol solution containing a mass concentration of 10%, and the mass ratio of the microspheres to the mannitol is 80:20 to 90:10 based on the solid content of the microsphere suspension;

[0025] The stirring speed was 50 rpm / min and the stirring time was 5 min.

[0026] According to the above description, mannitol solution can prevent microspheres from sticking together during freeze-drying, which results in decreased stability.

[0027] Optionally, in step S6, the specific steps of the freeze-drying treatment are: placing the microsphere suspension in a vacuum freeze dryer, first cooling it to -40°C, pre-freezing it for 1 hour, then heating it to -15°C, keeping it for 6 hours, then adjusting the temperature to -40°C, keeping it for 2 hours, turning on the vacuum pump, and after the vacuum degree is ≤10 Pa, gradually heating it to 0°C at a rate of 1°C per hour, keeping it for 20 hours, then gradually heating it to 40°C at a rate of 2°C per hour, keeping it for 20 hours, taking it out and passing it through a 65-mesh sieve, shaking it well, and obtaining the freeze-dried product of terlipressin sustained-release microspheres.

[0028] Another aspect of the present invention provides terlipressin sustained-release microspheres prepared by the above preparation method. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a scanning electron micrograph of the terlipressin sustained-release microspheres prepared in Example 1 of the present invention;

[0030] Figure 2 This is a particle size distribution diagram of terlipressin sustained-release microspheres prepared in Example 1 of the present invention;

[0031] Figure 3 This is a scanning electron micrograph of the terlipressin sustained-release microspheres prepared in Comparative Example 1 of the present invention;

[0032] Figure 4 This is a particle size distribution diagram of terlipressin sustained-release microspheres prepared in Comparative Example 1 of the present invention;

[0033] Figure 5 This is the in vitro cumulative release curve of the terlipressin sustained-release microspheres prepared in Example 1 of the present invention;

[0034] Figure 6 This is the in vivo blood drug concentration-time curve of SD rats injected with the terlipressin sustained-release microspheres prepared in Example 1. DETAILED DESCRIPTION

[0035] In order to better understand the above technical solution, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to enable a clearer and more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0036] This embodiment provides a method for preparing terlipressin sustained-release microspheres, comprising the following steps:

[0037] S1. Dissolve terlipressin acetate in pure water to prepare an inner aqueous phase.

[0038] The mass ratio of the terlipressin acetate to the pure water is 1:1 to 1:3.

[0039] In a preferred embodiment of the present invention, the mass ratio of the terlipressin acetate to the pure water is 1:2.

[0040] The method further comprises adding a protein protective agent into the inner aqueous phase, wherein the protein protective agent is at least one of sucrose, fructose, maltose and trehalose, and the concentration thereof is 10 to 30 mg / mL.

[0041] In a preferred embodiment of the present invention, the protein protective agent is sucrose, and its concentration is 15 mg / mL.

[0042] S2. dissolving the polylactic acid-glycolic acid copolymer in an organic solvent to obtain an oil phase.

[0043] The mass ratio of the polylactic acid-glycolic acid copolymer to the organic solvent is 1:2-1:3, the monomer ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is 50:50, and the organic solvent is dichloromethane.

[0044] In a preferred embodiment of the present invention, the mass ratio of the polylactic acid-glycolic acid copolymer to the organic solvent is 1:3.

[0045] S3. Dissolve polyvinyl alcohol in pure water and adjust the pH value to 7.8-8.5 to obtain an external water phase.

[0046] In a preferred embodiment of the present invention, the pH value is 8.2-8.3.

[0047] The external aqueous phase contains 0.1 wt% polyvinyl alcohol;

[0048] The pH value range is adjusted by a pH adjuster, and the pH adjuster is sodium bicarbonate and sodium hydroxide.

[0049] S4, adding the inner water phase into the oil phase for emulsification to obtain W / O colostrum.

[0050] The volume ratio of the inner water phase to the oil phase is 1:10 to 1:20;

[0051] The emulsification adopts a high-speed homogenization emulsification method, the emulsification temperature is 20-35° C., the emulsification speed is 15000 rpm, and the emulsification time is 10-20 minutes.

[0052] In a preferred embodiment of the present invention, the volume ratio of the inner water phase to the oil phase is 1:15; the emulsification temperature is 25° C., and the emulsification time is 20 min.

[0053] S5, mixing the colostrum with the external water phase, and performing secondary emulsification to obtain a W / O / W multiple emulsion.

[0054] The volume ratio of the colostrum to the external aqueous phase is 1:100 to 1:500;

[0055] The secondary emulsification adopts a high-speed homogenization emulsification method, and the secondary emulsification rotation speed is 10000-18000rpm.

[0056] In a preferred embodiment of the present invention, the volume ratio of the colostrum to the external aqueous phase is 1:300; and the secondary emulsification rotation speed is 15000 rpm.

[0057] S6. Stirring at room temperature to remove the organic solvent, collecting the microspheres by centrifugation, washing with pure water, and then freeze-drying to obtain the freeze-dried terlipressin sustained-release microspheres.

[0058] In a preferred embodiment of the present invention, the stirring time is 3-5 hours, preferably 5 hours; and the number of pure water washings is 2 times.

[0059] The method also includes, before the freeze-drying treatment, stirring and adding a freeze-drying protective agent to the microsphere concentrate obtained after washing to prepare a microsphere suspension, wherein the freeze-drying protective agent is a mannitol solution with a mass concentration of 10%, and the mass ratio of the microspheres to the mannitol is 80:20 to 90:10 based on the solid content of the microsphere suspension;

[0060] The stirring speed was 50 rpm / min and the stirring time was 5 min.

[0061] In a preferred embodiment of the present invention, based on the solid content of the microsphere suspension, the mass ratio of the microspheres to the mannitol is 85:15.

[0062] The specific steps of the freeze-drying treatment are: placing the microsphere suspension in a vacuum freeze dryer, first cooling it to -40°C, pre-freezing it for 1 hour, then heating it to -15°C, keeping it for 6 hours, then adjusting the temperature to -40°C, keeping it for 2 hours, turning on the vacuum pump, and after the vacuum degree is ≤10 Pa, gradually heating it to 0°C at a rate of 1°C per hour, keeping it for 20 hours, then gradually heating it to 40°C at a rate of 2°C per hour, keeping it for 20 hours, taking it out and passing it through a 65-mesh sieve, shaking it evenly, and obtaining the freeze-dried product of terlipressin sustained-release microspheres.

[0063] Example 1

[0064] A method for preparing terlipressin sustained-release microspheres comprises the following steps:

[0065] S1. Dissolve 1.0 g of terlipressin acetate in 2.0 g of pure water, and add 15 mg / mL sucrose as a protein protectant to prepare an inner aqueous phase.

[0066] S2. Dissolve 10.0 g of polylactic acid-glycolic acid copolymer (PLGA, wherein the monomer ratio of lactic acid to glycolic acid is 50:50) in 30.0 g of dichloromethane to prepare an oil phase.

[0067] S3. Dissolve 10.0 g of polyvinyl alcohol in 1000.0 g of pure water, and adjust the pH value to 8.2-8.3 using a pH adjuster (sodium bicarbonate and sodium hydroxide) to obtain an external aqueous phase.

[0068] S4. Add the inner water phase into the oil phase (the volume ratio of the inner water phase to the oil phase is 1:15), and emulsify by high-speed homogenization emulsification method, with an emulsification temperature of 25° C., an emulsification speed of 15000 rpm, and an emulsification time of 20 min to obtain W / O colostrum.

[0069] S5, mixing the colostrum with the external aqueous phase (the volume ratio of colostrum to external aqueous phase is 1:300), and performing secondary emulsification by high-speed homogenization emulsification method at a secondary emulsification speed of 15000 rpm to obtain a W / O / W double emulsion.

[0070] S6. Stir at room temperature for 5 hours to remove dichloromethane, collect the microspheres by centrifugation, wash twice with pure water, collect the washed microsphere concentrate, and stir to add a 10% mannitol solution as a lyophilization protectant. The stirring speed is 50 rpm / min and the stirring time is 5 minutes to prepare a microsphere suspension. The mass ratio of the microspheres to the mannitol is 85:15 based on the solid content of the microsphere suspension. The microspheres are then freeze-dried to obtain a finished freeze-dried product of terlipressin sustained-release microspheres.

[0071] Among them, the specific steps of the freeze-drying treatment are: placing the microsphere suspension in a vacuum freeze dryer, first cooling to -40°C, pre-freezing for 1 hour, then heating to -15°C, maintaining for 6 hours, then adjusting the temperature to -40°C, maintaining for 2 hours, turning on the vacuum pump, and after the vacuum degree is ≤10pa, gradually heating to 0°C at a rate of 1°C per hour, maintaining for 20 hours, and then gradually heating to 40°C at a rate of 2°C per hour, maintaining for 20 hours, taking out and passing through a 65-mesh sieve, shaking well, and obtaining the freeze-dried product of terlipressin sustained-release microspheres.

[0072] result: Figure 1 and 2 The scanning electron microscope image and particle size distribution diagram of the terlipressin sustained-release microspheres prepared according to the present invention in this embodiment are shown respectively. 50 The particle size was 12.3 μm, the diameter distance was 2.888, the drug loading was 7.8%, the encapsulation efficiency was 84.6%, and the yield of terlipressin was 88.3%.

[0073] Example 2

[0074] The difference between this embodiment and embodiment 1 is that the emulsification time for preparing colostrum is different (10 min).

[0075] Example 3

[0076] The difference between this embodiment and embodiment 1 is that the emulsification time for preparing colostrum is different (15 min).

[0077] Example 4

[0078] The difference between this embodiment and embodiment 1 is that the emulsification speed for preparing the double emulsion is different (10000 rpm).

[0079] Example 5

[0080] The difference between this embodiment and embodiment 1 is that the emulsification speed for preparing the double emulsion is different (12000 rpm).

[0081] Example 6

[0082] The difference between this embodiment and embodiment 1 is that the emulsification speed for preparing the double emulsion is different (18000 rpm).

[0083] Comparative Example 1

[0084] The main difference between this comparative example and Example 1 is that no protein protective agent is added in step S1; in step S3, the pH value of the external water phase is adjusted to 5.2-5.6. Specifically, the following steps are included:

[0085] S1. Dissolve 1.0 g of terlipressin acetate in 1.5 g of pure water to prepare an inner aqueous phase.

[0086] S2. Dissolve 12.0 g of polylactic acid-glycolic acid copolymer (wherein the monomer ratio of lactic acid to glycolic acid is 50:50) in 24.0 g of dichloromethane to obtain an oil phase.

[0087] S3. Dissolve 10.0 g of polyvinyl alcohol in 1000.0 g of pure water, and adjust the pH value to 5.2-5.6 using a pH adjuster (acetic acid and sodium acetate) to obtain an external water phase.

[0088] S4. Add the inner water phase into the oil phase (the volume ratio of the inner water phase to the oil phase is 1:15), and emulsify by high-speed homogenization emulsification method, with an emulsification temperature of 30° C., an emulsification speed of 15000 rpm, and an emulsification time of 15 min to obtain W / O colostrum.

[0089] S5, mixing the colostrum with the external aqueous phase (the volume ratio of colostrum to external aqueous phase is 1:300), and performing secondary emulsification by high-speed homogenization emulsification method at a secondary emulsification speed of 15000 rpm to obtain a W / O / W double emulsion.

[0090] S6. Stir at room temperature for 3 hours to remove dichloromethane, collect the microspheres by centrifugation, wash twice with pure water, collect the washed microsphere concentrate, and stir to add a 10% mannitol solution as a lyophilization protectant. The stirring speed is 50 rpm / min and the stirring time is 5 minutes to prepare a microsphere suspension. The mass ratio of the microspheres to the mannitol is 90:10 based on the solid content of the microsphere suspension. The microspheres are then freeze-dried to obtain a finished freeze-dried product of terlipressin sustained-release microspheres.

[0091] Among them, the specific steps of the freeze-drying treatment are: placing the microsphere suspension in a vacuum freeze dryer, first cooling to -40°C, pre-freezing for 1 hour, then heating to -15°C, maintaining for 6 hours, then adjusting the temperature to -40°C, maintaining for 2 hours, turning on the vacuum pump, and after the vacuum degree is ≤10pa, gradually heating to 0°C at a rate of 1°C per hour, maintaining for 20 hours, and then gradually heating to 40°C at a rate of 2°C per hour, maintaining for 20 hours, taking out and passing through a 65-mesh sieve, shaking well, and obtaining the freeze-dried product of terlipressin sustained-release microspheres.

[0092] result: Figure 3 and 4 The scanning electron microscope image and particle size distribution diagram of the terlipressin sustained-release microspheres prepared in this comparative example are shown respectively. 50 The particle size was 11.8 μm, the line spacing was 3.281, the drug loading was 6.8%, the encapsulation efficiency was 51.9%, and the yield of terlipressin was 55.7%.

[0093] Comparative Example 2

[0094] The difference between this comparative example and Example 1 is that the pH adjustment of the inner water phase and the outer water phase is different (adjusted by sodium bicarbonate / sodium hydroxide and acetic acid / sodium acetate), and the specific adjustment is shown in Table 1.

[0095] Table 1

[0096] serial number pH value of internal water phase External water phase pH <![CDATA[1 # ]]> Unregulated 5.2～5.6 <![CDATA[2 # ]]> Unregulated 10.2～10.3 <![CDATA[3 # ]]> 5.2～5.6 Unregulated <![CDATA[4 # ]]> 8.2～8.3 Unregulated <![CDATA[5 # ]]> 10.2～10.3 Unregulated

[0097] Comparative Example 3

[0098] The difference between this comparative example and Example 1 is that the process of step S6 is different. The specific adjustments are as follows:

[0099] The mixture was stirred at room temperature for 3 h to remove dichloromethane, and the microspheres were collected by centrifugation and washed twice with pure water. The washed microsphere concentrate was collected and stirred to add a 10% mannitol solution as a freeze-drying protective agent. The stirring speed was 50 rpm / min and the stirring time was 5 min to prepare a microsphere suspension. The mass ratio of the microspheres to the mannitol was 80:20 based on the solid content of the microsphere suspension. The mixture was then freeze-dried to obtain a finished freeze-dried product of terlipressin sustained-release microspheres.

[0100] The specific steps of the freeze-drying treatment are as follows: placing the microsphere suspension in a vacuum freeze dryer, adjusting the temperature to -40°C, maintaining for 3 hours, turning on the vacuum pump, and after the vacuum degree is ≤10pa, gradually heating to 0°C at a rate of 1°C per hour, maintaining for 20 hours, and then gradually heating to 40°C at a rate of 2°C per hour, maintaining for 20 hours, taking out and passing through a 65-mesh sieve, shaking well, and obtaining the freeze-dried product of terlipressin sustained-release microspheres.

[0101] Comparative Example 4

[0102] The difference between this comparative example and Example 1 is that the freeze-drying process in step S6 is different. The specific adjustments are as follows:

[0103] The specific steps of the freeze-drying treatment are: placing the microsphere suspension in a vacuum freeze dryer, adjusting the temperature to -40°C, maintaining for 3 hours, turning on the vacuum pump, and after the vacuum degree is ≤10 Pa, gradually heating to 0°C at a rate of 1°C per hour, maintaining for 20 hours, and then gradually heating to 40°C at a rate of 2°C per hour, maintaining for 20 hours, taking out and passing through a 65-mesh sieve, shaking well, and obtaining the finished freeze-dried terlipressin sustained-release microspheres.

[0104] Performance Testing

[0105] (I) The terlipressin sustained-release microspheres prepared in Example 1 were subjected to an in vitro release test.

[0106] The specific method is as follows:

[0107] 1. Preparation of dissolution medium: weigh 34 g of Tris base, 3 g of Tris HCl, 5 g of poloxamer (188), and 0.2 g of sodium azide, add 1000 mL of water to dissolve, and adjust the pH to 7.4 ± 0.1.

[0108] 2. Preparation of sample solution: Take the lyophilized product of terlipressin sustained-release microspheres prepared according to Example 1, accurately weigh 89.3 mg (equivalent to 10.0 mg of the raw material terlipressin) and place it in a pre-treated dialysis bag, add 2 mL of dissolution medium, and tie the bag tightly.

[0109] 3. Collection of dialysate: Place the dialysis bag in a container containing 400 mL of dissolution medium, and oscillate it in a constant temperature oscillator, set the temperature to 37°C and the speed to 100 rpm. Take out 10 mL of dialysate at regular intervals at 3h, 6h, 12h, 24h, 36h, 48h, and 96h, and replenish an equal amount of fresh dissolution medium in time.

[0110] 4. Content determination: After filtering the sample solutions at different time points through a 0.22μm microporous filter membrane, take the subsequent filtrate as the test solution. According to the method under the content determination item in the 2015 edition of the Pharmacopoeia, the content of terlipressin acetate in the dissolution medium is determined by high performance liquid chromatography. Take an appropriate amount of terlipressin acetate reference substance, accurately weigh it, dissolve it in water and dilute it to make a solution containing approximately 0.4mg per 1mL, and determine it in the same way. Calculate the drug concentration in the test sample by peak area according to the external standard method, calculate the cumulative release, and draw a cumulative release-time relationship curve (see Figure 5 ).

[0111] Results: From Figure 5 It can be seen that the cumulative release of the microspheres at 3h, 24h, and 48h are 12.2%, 60.7%, and 95.9%, respectively, which indicates that the microspheres can achieve a stable release of 48h and release the drug completely, which is sufficient to maintain the effect of the drug.

[0112] (II) The terlipressin sustained-release microspheres prepared in Example 1 were subjected to in vivo pharmacokinetic study in rats. The specific method is as follows:

[0113] 1. Preparation of drug solution:

[0114] Terlipressin sustained-release microsphere solution: Take the dry product of terlipressin sustained-release microspheres prepared in Example 1, weigh it accurately, dissolve it in physiological saline and quantitatively dilute it to make 1 mg of terlipressin per 1 mL.

[0115] Terlipressin solution for injection: Take commercially available lyophilized powder of terlipressin for injection, accurately weigh it, dissolve it in physiological saline and quantitatively dilute it to make 1 mg of terlipressin per 1 mL.

[0116] 2. Experimental animals and groups: Sixteen SD rats aged 6-8 weeks, half male and half female, were randomly divided into 2 groups, with 8 rats in each group.

[0117] 3. Administration: A single intravenous injection of 50 μg / kg body weight was given, respectively, as terlipressin sustained-release microsphere solution and terlipressin injection solution.

[0118] 4. Collection and processing of plasma samples: 100 μL of blood was collected from the eye sockets before administration and at 5 minutes, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 10 hours, 24 hours, and 72 hours after administration. The blood was anticoagulated with 1% heparin and centrifuged at 8000×g for 5 minutes to separate the plasma. It was stored at -80℃ until testing.

[0119] 5. Content determination: Take plasma samples at different time points and put them in a centrifuge tube, add 200μL of acetonitrile solution, vortex for 60s, centrifuge at 1500×g for 10min at 4℃, and take 20μL of the supernatant for injection. According to the method under the content determination item in the 2015 edition of the Pharmacopoeia, the content of terlipressin acetate was determined by high performance liquid chromatography. Take an appropriate amount of terlipressin acetate reference substance, accurately weigh it, dissolve it in water and dilute it to make a solution containing approximately 0.4mg per 1ml, and determine it in the same way. Calculate the drug concentration in the test sample by peak area using the external standard method, and draw a blood drug concentration-time curve (see Figure 6 ).

[0120] Results: For intravenous terlipressin sustained-release microsphere solution, the average plasma concentration decreased slowly after administration, and the average plasma concentrations at 10h, 24h and 72h were 3.55ng / mL, 1.25ng / mL, 0.50ng / mL, respectively, with a half-life of 18.5h; for injection terlipressin solution, the average plasma concentrations at 1h and 2h after administration were 2.40ng / mL and 0.35ng / mL, respectively, with a half-life of 19min. The above results show that the terlipressin sustained-release microspheres of the present invention can achieve slow release.

[0121] (III) The terlipressin sustained-release microspheres prepared in Example 1 and Comparative Example 2 were tested, and the results are recorded in Table 2.

[0122] Table 2

[0123] serial number pH value of internal water phase External water phase pH <![CDATA[Microsphere particle size (D 50 , μm)]]> Encapsulation rate (%) <![CDATA[1 # ]]> Unregulated 5.2～5.6 11.8 51.9 Example 1 Unregulated 8.2～8.3 12.3 84.6 <![CDATA[2 # ]]> Unregulated 10.2～10.3 27.9 63.4 <![CDATA[3 # ]]> 5.2～5.6 Unregulated 12.7 76.7 <![CDATA[4 # ]]> 8.2～8.3 Unregulated 31.2 8.2 <![CDATA[5 # ]]> 10.2～10.3 Unregulated 28.9 10.5

[0124] Results: When the pH value of the external aqueous phase was adjusted to 8.2-8.3, the microsphere encapsulation efficiency was 84.6%; when the pH value of the external aqueous phase was adjusted to 10.2-10.3, the encapsulation efficiency was slightly reduced to 63.4%. This may be due to the alkaline isoelectric point of terlipressin (about 8.2-8.3). When the pH value of the external aqueous phase is close to the isoelectric point, the solubility is the lowest, thereby reducing the migration of terlipressin from the internal aqueous phase to the external aqueous phase. Therefore, the encapsulation efficiency of the microspheres finally prepared is higher. And when the pH value of the external aqueous phase is 10.2-10.3, the particle size increases, D 50 The particle size of the microspheres is 27.9 μm, which may be due to the strong alkalinity of the external aqueous phase, which makes the properties of terlipressin unstable and the particle size distribution of the formed microspheres is uneven.

[0125] The inner aqueous phase is a terlipressin acetate aqueous solution, and the pH of the solution is measured to be in the range of 5.2 to 5.6. When the pH value of the inner aqueous phase is adjusted to 8.2 to 8.3, a small amount of precipitation can be seen during the solution preparation process, and the particle size distribution of the obtained microspheres is uneven. The measured D 50 The value was 31.2 μm, and the encapsulation efficiency was 8.2%. When the pH value of the inner aqueous phase was adjusted to 10.2-10.3, the microsphere encapsulation efficiency was 10.5%. The results showed that the change of the pH value of the inner aqueous phase would reduce the stability of terlipressin, resulting in an increase in the particle size of the final microsphere preparation and a decrease in the encapsulation efficiency.

[0126] (IV) The terlipressin sustained-release microspheres prepared in Examples 1-3 were tested, and the results are recorded in Table 3.

[0127] Table 3

[0128]

[0129]

[0130] As shown in Table 3, during the preparation of microspheres, the colostrum emulsification time had almost no effect on the particle size of the final microsphere preparation (D 50 ), but the change in colostrum particle size will affect the in vitro release. When the emulsification time is 20 min, the colostrum particle size (D 50 ) is 545nm, and the in vitro release experiment shows that the cumulative release rate within 48h is 95.9%.

[0131] (V) The terlipressin sustained-release microspheres prepared in Example 1 and Examples 4-6 were tested, and the results are recorded in Table 4.

[0132] Table 4

[0133] serial number Emulsion speed (rpm) <![CDATA[Microsphere particle size (D 50 , μm)]]> Microsphere drug loading (%) Example 4 10000 22.65 6.77 Example 5 12000 15.54 6.87 Example 1 15000 12.30 7.80 Example 6 18000 10.82 7.62

[0134] As can be seen from Table 4, the rotation speed of the emulsion during microsphere preparation has a great influence on the particle size of the final microsphere preparation. When the rotation speed is increased to 12000rpm, the microsphere particle size is significantly reduced compared with 10000rpm; when the rotation speed is 15000rpm, the microsphere particle size is reduced to 12.3μm; when the rotation speed is further increased to 18000rpm, the microsphere particle size does not decrease significantly. Considering the microsphere particle size and drug loading, the rotation speed of 15000rpm is finally determined to be the best.

[0135] (VI) Determination of dichloromethane content in the terlipressin sustained-release microspheres prepared in Example 1 and Comparative Examples 3-4: The dichloromethane content in the freeze-dried microspheres was determined by headspace gas chromatography. The results are recorded in Table 5.

[0136] Table 5

[0137]

[0138]

[0139] Dichloromethane is used as the oil phase solvent in the preparation process of microsphere preparations, and there may be problems with failure to completely remove it during the solidification process. Dichloromethane is an organic solvent with certain toxicity. Residual dichloromethane not only affects the stability of microspheres during storage, but also causes adverse reactions in the human body after injection. Therefore, quality control of dichloromethane is crucial to improving the stability and safety of microsphere preparations. Since the prepared terlipressin is a polypeptide drug that is unstable to heat, it is not suitable to remove dichloromethane by high-temperature stirring solvent volatilization.

[0140] As can be seen from Table 5, the present invention can reduce the residual amount of dichloromethane in the microsphere preparation by adjusting the temperature program of the freeze-drying process and introducing an annealing program.

[0141] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing terlipressin sustained-release microspheres, comprising the following steps: S1, dissolving terlipressin acetate in pure water to prepare an inner water phase; S2, dissolving the polylactic acid-glycolic acid copolymer in an organic solvent to obtain an oil phase; S3, dissolving polyvinyl alcohol in pure water and adjusting the pH value to 7.8-8.5 to prepare an external water phase; S4, adding the inner water phase to the oil phase for emulsification, the emulsification time is 10 to 20 minutes, to obtain W / O colostrum; S5, mixing the colostrum with the external water phase, performing secondary emulsification, the secondary emulsification speed is 10000-18000 rpm, to obtain a W / O / W double emulsion; S6, stirring at room temperature to remove the organic solvent, collecting the microspheres by centrifugation, washing with pure water, and then freeze-drying to obtain a freeze-dried product of terlipressin sustained-release microspheres; Step S1 further includes adding a protein protective agent to the inner aqueous phase, wherein the protein protective agent is at least one of sucrose, fructose, maltose and trehalose, and the concentration thereof is 10 to 30 mg / mL; In step S6, the specific steps of the freeze-drying treatment are: placing in a vacuum freeze dryer, first cooling to -40°C, pre-freezing for 1 hour, then heating to -15°C, maintaining for 6 hours, then adjusting the temperature to -40°C, maintaining for 2 hours, turning on the vacuum pump, and after the vacuum degree is ≤10pa, gradually heating to 0°C at a rate of 1°C per hour, maintaining for 20 hours, and then gradually heating to 40°C at a rate of 2°C per hour, maintaining for 20 hours, taking out and passing through a 65-mesh sieve, shaking well, and obtaining the freeze-dried product of terlipressin sustained-release microspheres.

2. The method for preparing terlipressin sustained-release microspheres according to claim 1, characterized in that: In step S1, the mass ratio of the terlipressin acetate to the pure water is 1:1 to 1:

3.

3. The method for preparing terlipressin sustained-release microspheres according to claim 1, characterized in that: In step S2, the mass ratio of the polylactic acid-glycolic acid copolymer to the organic solvent is 1:2-1:3, the monomer ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is 50:50, and the organic solvent is dichloromethane.

4. The method for preparing terlipressin sustained-release microspheres according to claim 1, characterized in that: In step S3, the external aqueous phase contains 0.1 wt % polyvinyl alcohol; The pH value range is adjusted by a pH adjuster, and the pH adjuster is sodium bicarbonate and sodium hydroxide.

5. The method for preparing terlipressin sustained-release microspheres according to claim 1, characterized in that: In step S4, the volume ratio of the inner water phase to the oil phase is 1:10 to 1:20; The emulsification adopts a high-speed homogenization emulsification method, the emulsification temperature is 20-35° C., and the emulsification speed is 15000 rpm.

6. The method for preparing terlipressin sustained-release microspheres according to claim 1, characterized in that: In step S5, the volume ratio of the colostrum to the external aqueous phase is 1:100 to 1:500; The secondary emulsification adopts a high-speed homogenization emulsification method.

7. The method for preparing terlipressin sustained-release microspheres according to claim 1, characterized in that: Step S6 further includes, before the freeze-drying treatment, stirring and adding a freeze-drying protective agent to the microsphere concentrate obtained after washing to prepare a microsphere suspension, wherein the freeze-drying protective agent is a mannitol solution containing a mass concentration of 10%, and the mass ratio of the microspheres to the mannitol is 80:20 to 90:10 based on the solid content of the microsphere suspension; The stirring speed was 50 rpm / min and the stirring time was 5 min.

8. Terlipressin sustained-release microspheres prepared by the preparation method according to any one of claims 1 to 7.

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